The present invention relates to the alleviation of hypotension induced by nitrogen oxide production. Research relating to the present invention was supported by government research grants which gives the United States government rights in the present invention.
In 1980, Furchgott and Zawadski (Nature 288:373-376 1980) demonstrated that endothelial cells, which line blood vessels, can be stimulated to release a substance which relaxes vascular smooth muscle i.e., causes vasodilatation. Since the chemical nature of this substance was completely unknown, it was simply named endothelium-derived relaxing factor (EDRF). It is now widely accepted that many naturally-occurring substances which act as physiological vasodilators mediate all or part of their action by stimulating release of EDRF; these substances include acetylcholine, histamine, bradykinin, leukotrienes, ADP, ATF, substance P, serotonin, thrombin and others. Although the extremely short lifetime of EDRF (several seconds) hampered efforts to chemically identify this molecule, in 1987 several laboratories suggested that EDRF may be nitric oxide (NO.), which spontaneously decomposes to nitrate and nitrite. A fundamental problem in accepting this No. hypothesis was that mammalian systems were not known to contain an enzymatic pathway which could synthesize NO.; additionally, a likely precursor for NO. biosynthesis was unknown. After observing that the arginine analog N.sup.G -methyl-L-arginine (L-NMA) could inhibit vascular EDRF/NO. synthesis induced by acetylcholine and histamine, and that EDRF/NO. synthesis could be restored by adding excess L-arginine, it was proposed that arginine is the physiological precursor of EDRF/NO. biosynthesis (Sakuma et al., PNAS 85:8664-8667, 1988). Additional evidence supporting this proposal was reported almost simultaneously. It was also later demonstrated that inhibition of EDRF/NO. synthesis in the anesthetized guinea pig raises blood pressure, suggesting that EDRF/NO. is an important physiological regulator of blood pressure (Aisaka et al., BBRC 160:881-886, 1989). Notwithstanding the accumulated evidence supporting synthesis of NO., it is understood by those skilled in the art that other nitrogen oxides may be present and may be active in reducing blood pressure. Within this specification, the acronym NO. will be understood to represent nitric oxide and any additional vasoactive nitrogen oxides.
It was demonstrated that macrophage cells become "activated" by 12-36 hour treatment with gamma-interferon, bacterial endotoxin and various cytokines. This "activation" is associated with initiation of tumor cell killing and generation of nitrite and nitrate from L-arginine. It was also observed that activated macrophages actually make NO. from L-arginine (just like endothelial cells) and that this NO. subsequently reacts with oxygen to form more oxidized nitrogen metabolites which appear to be physiologically inert (Stuehr et al., J. Exp. Med. 169:1011-1020, 1989). The enzyme responsible for NO. synthesis (nitric oxide synthase) has been partially characterized by some of the present inventors (Stuehr et al. BBRC 161:420-426, 1989) and acts to oxidize the terminal amino group of arginine, resulting in production of NO. and citrulline. It is now believed that macrophage-derived NO. is an important tumoricidal and bactericidal agent. Since bacterial endotoxin, gamma-interferon and other cytokines can trigger NO. generation by macrophage cells it appeared that: 1) endothelial cell NO. generation may be stimulated by similar stimuli and 2) septic shock (i.e., systemic vasodilatation induced by bacterial endotoxin) may result from massive activation of NO. biosynthesis. Speculation that the latter hypothesis was correct was fueled by a prior report that urinary nitrate levels are grossly elevated by treatment of rats with bacterial endotoxin (Wagner et al., PNAS 80:4518-4521, 1983).
Cytokines are well known to cause morphological and functional alterations in endothelial cells described as "endothelial cell activation". Distinct immune-mediators such as tumor necrosis factor (TNF), interleukin-1 (IL-1), and gamma-interferon (IFN or I) appear to induce different but partially overlapping patterns of endothelial cell activation including increased procoagulant activity (Bevilaqui et al., PNAS 83:4533-4537, 1986), PGI2 production (Rossi, Science 229,174, 1985), HLA antigen expression (Pober et al., J. Immunol. 138:3319-3324, 1987) and lymphocyte adhesion molecules (Cavender et al., J. Immunol. 138:2149-2154, 1987). Although these cytokines are reported to cause hypotension, vascular hemorrhage, and ischemia, the underlying mechanisms of altered vasoactivity are unclear (Goldblum et al., Infect. Immun. 57:1218-1226, 1989; Tracey et al., Science 234:470, 1986). A potential mediator of altered vasoactivity is EDRF.
In both clinical and animal (Dvorak, et al., J. Nat'l. Canc. Inst. 81:497-502, 1959) studies on the effects of biological response modifiers a major dose limiting toxicity has been hypotension and vascular leakage.
The cardiotonic drug dobutamine has been used as an addition to a standard treatment protocol, to treat patients in septic shock. Results obtained indicated that dobutamine did not significantly increase mean arterial pressure but did increase oxygen supply and consumption (Vincent et al., 1980, Critical Care Medicine 18:689-693).
Kilbourn et al. described a decrease in blood pressure and cardiac output in dogs treated with endotoxin or IL-1 (BBRC 178:823-829, 1991; JNCI 1992 in press). The administration of N.sup.G -methyl-L-arginine (NMA) partially restored blood pressure but further decreased cardiac output. This result was recently confirmed by Klabunde et al. (Klabunde BBRC 178:1135-1140, 1991). Further studies showed that NMA decreases cardiac output in normal animals (not treated with endotoxin) an effect which is increased at higher doses of NMA (Kilbourn et al. BBRC 178:823-829, 1991; Klabunde et al. Eur. J. Pharm. 199:51-59, 1991).